CN107075731A - 一种核酸单链环状文库的构建方法和试剂 - Google Patents
一种核酸单链环状文库的构建方法和试剂 Download PDFInfo
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- CN107075731A CN107075731A CN201480081852.6A CN201480081852A CN107075731A CN 107075731 A CN107075731 A CN 107075731A CN 201480081852 A CN201480081852 A CN 201480081852A CN 107075731 A CN107075731 A CN 107075731A
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Abstract
Description
组分 | 含量 |
转座酶 | 85μL |
一号接头 | 30μL |
偶联缓冲液 | 85μL |
合计 | 200μL |
组分 | 含量 |
水 | 5μL |
5×打断缓冲液 | 2μL |
gDNA(50ng/μL) | 1μL |
打断酶复合体 | 2μL |
合计 | 10μL |
组分 | 含量 |
水 | 8μL |
3×连接buffer | 20μL |
二号接头(5μM) | 10μL |
连接酶 | 2μL |
DNA | 20μL |
合计 | 30μL |
组分 | 含量 |
DNA | 30μL |
5×PCR缓冲液 | 10μL |
10mM dNTPs | 1μL |
标签引物1-8 | 2μL |
引物2 | 2μL |
PCR酶 | 1μL |
纯水 | 4μL |
合计 | 50μL |
组分 | 含量 |
DNA | 120μL |
5×PCR缓冲液 | 40μL |
10mM dNTPs | 4μL |
通用引物1 | 8μL |
生物素引物2 | 8μL |
PCR酶 | 4μL |
纯水 | 16μL |
合计 | 200μL |
组分 | 含量 |
“桥”序列 | 20μL |
纯水 | 178.3μL |
10×连接缓冲液 | 35μL |
100mM ATP | 3.5μL |
连接酶 | 1.2μL |
上一步回收的单链DNA | 112μL |
合计 | 350μL |
组分 | 含量 |
10×连接缓冲液 | 3.7μL |
20U/μL外切酶I | 11.1μL |
100U/μL外切酶III | 5.2μL |
合计 | 20μL |
参数 | 数值 |
浓度 | 1.62ng/μL |
体积 | 40μL |
总量 | 64.8ng |
摩尔数 | 0.65pmol |
参数 | 数值 |
覆盖度 | 98.6% |
SNP数目 | 42000 |
dbSNP数据一致性 | 98.65% |
基因分型一致性 | 99.97% |
Claims (14)
- 一种核酸单链环状文库的构建方法,包括如下步骤:使用转座酶包埋复合体对核酸进行随机打断,其中所述转座酶包埋复合体包含转座酶和含转座酶识别序列的第一接头,打断的核酸两端连接所述第一接头并且形成缺口;去除体系中的转座酶,然后使用连接酶在所述缺口处连接上第二接头,所述第二接头的序列不同于所述第一接头;使用分别靶向结合所述第一接头和所述第二接头的引物进行第一PCR反应,得到两端分别连接有不同接头序列的产物,其中一条引物在5’端具有第一亲和标记;将所述第一PCR反应的产物与具有第二亲和标记的固相载体接触,使所述第一亲和标记与所述第二亲和标记形成亲和结合;对结合到所述固相载体上的PCR产物进行变性处理,分离出无亲和标记的单链;使用单链环化“桥”序列对所述单链进行环化,其中所述单链环化“桥”序列能够同时结合所述单链的两端。
- 根据权利要求1所述的方法,其特征在于,在所述第一PCR反应之前,使用分别靶向结合所述第一接头和所述第二接头的引物进行第二PCR反应,得到两端分别连接有不同接头序列的产物。
- 根据权利要求2所述的方法,其特征在于,所述第二PCR反应使用的引物对中有一条引物在5’端具有样本标签序列。
- 根据权利要求2或3所述的方法,其特征在于,在所述第二PCR反应之后且在所述第一PCR反应之前,使用外显子序列探针捕获所述第二PCR反应的产物中含有外显子序列的单链,其中所述外显子序列探针具有所述第一亲和标记,能够与所述固相载体上的第二亲和标记形成亲和结合,而将所述含有外显子序列的单链分离出来,以用于进行第一PCR反应。
- 根据权利要求4所述的方法,其特征在于,在使用外显子序列探针捕获所述第二PCR反应的产物中含有外显子序列的单链之前,使用引物封闭序列封闭所述第二PCR反应得到的产物单链两端的引物序列。
- 根据权利要求1所述的方法,其特征在于,所述第一亲和标记为生物素标记;所述第二亲和标记为链霉亲和素标记。
- 根据权利要求1所述的方法,其特征在于,所述去除体系中的转座酶采用磁珠纯化、过柱纯化或化学试剂处理的方式进行。
- 根据权利要求1所述的方法,其特征在于,所述固相载体为磁珠。
- 根据权利要求1所述的方法,其特征在于,所述方法包括如下步骤:使用转座酶包埋复合体对核酸进行随机打断,其中所述转座酶包埋复合体包含转座酶和含转座酶识别序列的第一接头,打断的核酸两端连接所述第一接头并且形成缺口;去除体系中的转座酶,然后使用连接酶在所述缺口处连接上第二接头,所述第二接头的序列不同于所述第一接头;使用分别靶向结合所述第一接头和所述第二接头的引物进行第二PCR反应,得到两端分别连接有不同接头序列的产物,其中一条引物在5’端具有样本标签序列;使用引物封闭序列封闭所述第二PCR反应得到的产物单链两端的引物序列;使用外显子序列探针捕获所述第二PCR反应的产物中含有外显子序列的单链,其中所述外显子序列探针具有生物素标记,能够与固相载体上的链霉亲和素标记形成亲和结合,而将所述含有外显子序列的单链分离出来;使用针对所述含有外显子序列的单链两端的引物进行第一PCR反应,得到两端分别连接有不同接头序列的产物,其中一条引物在5’端具有生物素标记;将所述第一PCR反应的产物与具有链霉亲和素标记的固相载体接触,使所述生物素标记与所述链霉亲和素标记形成亲和结合;对结合到所述固相载体上的PCR产物进行变性处理,分离出无亲和标记的单链;使用单链环化“桥”序列对所述单链进行环化,其中所述单链环化“桥”序列能够同时结合所述无亲和标记的单链的两端。
- 一种核酸单链环状文库的构建试剂,包括如下组成部分:转座酶和含转座酶识别序列的第一接头,用于形成转座酶包埋复合体以对核酸进行随机打断,在打断的核酸两端连接所述第一接头并且形成缺口;第二接头和连接酶组分,用于在所述缺口处连接上第二接头;用于第一PCR反应的引物,其中一条引物在5’端具有第一亲和标记,所述 引物分别靶向结合所述第一接头和所述第二接头;固相载体,具有第二亲和标记,用于与所述第一亲和标记形成亲和结合;变性溶液,用于对结合到所述固相载体上的PCR产物进行变性处理,以分离出无亲和标记的单链;单链环化“桥”序列,能够同时结合所述单链的两端,用于对所述单链进行环化。
- 根据权利要求10所述的试剂,其特征在于,所述试剂还包括用于第二PCR反应的引物,用于分别靶向结合所述第一接头和所述第二接头;优选地,所述用于第二PCR反应的引物中有一条引物在5’端具有样本标签序列。
- 根据权利要求10或11所述的试剂,其特征在于,所述试剂还包括外显子序列探针,用于捕获所述第二PCR反应的产物中含有外显子序列的单链,所述外显子序列探针具有所述第一亲和标记,能够与所述固相载体上的第二亲和标记形成亲和结合,而将所述含有外显子序列的单链分离出来;优选地,所述试剂还包括引物封闭序列,用于封闭所述第二PCR反应得到的产物单链两端的引物序列。
- 根据权利要求10所述的试剂,其特征在于,所述第一亲和标记为生物素标记;所述第二亲和标记为链霉亲和素标记。
- 根据权利要求10所述的试剂,其特征在于,所述固相载体为磁珠。
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